This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To define the differentiation potential of human induced pluripotent stem cells. The formation of the trophectoderm lineage represents one of the earliest events in mammalian embryonic differentiation. Pluripotent stem cells derived from human and animal embryos exhibit a range of differentiation potential in their ability to form the trophoblast lineage upon spontaneous or directed differentiation. These observations may reflect the heterogeneous conditions and events that lead to the immortalization of embryonic cells to embryonic stem cells. Induced pluripotent stem cells (iPSC) provide an opportunity to shed further light on the requirements for trophoblast formation in human embryos. The human iPSC lines IMR90-1, -2, and Foreskin-1, -2, and -3 were subjected to in vitro culture conditions shown to induce trophoblast differentiation in human ESC: treatment with BMP-4, and embryoid body formation followed by adherent culture on 2-dimensional or 3-dimensional Matrigel environments. Trophoblast differentiation was monitored by secretion of human chorionic gonadotropin (hCG), and by histological and immunohistochemical analysis of embryoid bodies formed from iPSC. In all cell lines, treatment with BMP-4 but not activin, TGF-beta, FGF4, or zebrafish FGF2 resulted in a dose-related increase in the secretion of hCG into the culture medium within 6 days of treatment. Likewise, adherent culture of embryoid bodies on Matrigel-coated 2-dimensional surfaces supported consistent initiation of hCG expression during up to 4 weeks of culture. Somewhat unexpectedly, iPSC-derived embryoid bodies less consistently upregulated the secretion of hCG when placed into 3-dimensional Matrigel explants. This was in agreement with difficulty in detecting hCG secretion by immunohistochemistry in these 3-D embryoid body cultures, although trophoblast differentiation was suggested by cytokeratin staining of surface and outgrowth cells. We conclude that iPSC have similar responses to BMP-4 treatment as human embryo-derived pluripotent stem cells with regard to trophoblast differentiation, and that further evaluation of differentiation in 3-dimensional extracellular matrix environments is needed. This research used WNPRC Stem Cell Resources.